Ethyl 3-[[bis(1-methylethoxy)phosphinothioyl]thio]propionate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-04-17 to 2014-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to internationally accepted guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the start of the exposure intervals (0 and 24 h), sampling was carried out immediately after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 h), samples were taken directly from the test vessels. The concentrations of the test were analytically verified at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control.

- Preparation of the standards: A stock solution of 1000 mg test item/L in acetonitrile was diluted to 10 mg/L with acetonitrile: HPLC water (50 : 50) containing 0.1 % formic acid. This solution was further diluted with acetonitrile: HPLC water (50 : 50) containing 0.1 % formic acid and used for calibration (8 concentrations).

- Preparation of the samples: The samples and the control were stabilized (dilution factor 2) with acetonitrile containing 0.2 % formic acid. Further dilutions were made prior to analysis with acetonitrile: HPLC water (50 : 50) containing 0.1 % formic acid.

- Sample storage conditions before analysis: All samples were stored at room temperature until sample preparation and until start of analysis.

Vehicle:

no

Details on test solutions:

STOCK SOLUTION
The stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h).

PREPARATION AND APPLICATION OF TEST SOLUTION
The stock solution of 10.0 mg/L was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h) and treated with ultrasound for 10 minutes at room temperature.
Further test concentrations were prepared by diluting the stock solution of 10.0 mg/L with dilution water. The tested solutions were clear throughout the exposure period. At the end of the first exposure interval (after 24 h), a slightly reddish sediment was observed at the concentration levels of 5.00 and 10.0 mg/L.

TREATMENT:
The stock solution was treated with ultrasound for 10 minutes at room temperature.

DILUTION WATER: Same as culture medium: Elend M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
- Age of the daphnids at the start of the exposure: 2 to 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test 2 hours after separation from the culture vessels. No first brood progeny was used for the test.
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Breeding conditions: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium,
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Temperature: 20 ± 2 °C in an incubator
- Photoperiod: 16 h illumination
- Light Intensity: Max. 20 µE/m2/s
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 1000000 cells/mL
- Feeding: The daphnids were not fed during the study.

ACCLIMATION
Acclimatization was not necessary, because the dilution water and environmental conditions were equivalent to the culture.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

168 - 176 mg CaCO3/L

Test temperature:

21.9 - 22 °C

pH:

8.22 -7.48

Dissolved oxygen:

8.37 - 8.58 mg/L

Salinity:

-Conductivity: 474 -478 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 5 test item concentrations in a geometric series with a separation factor of 2 were tested as follows: 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L.
- Measured concentrations: The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 80 -to 106 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 72 to 96 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be: 0.500, 1.07, 2.15, 4.66 and 9.85 mg/L.
- Control: Dilution water without test item tested under the same conditions as the test groups.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with glasses
- Test volume: 20 mL
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Aeration: yes
- Application: Per replicate, 20 g test solution were weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline, same as culture medium
- The water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured at the start of the exposure intervals (0 and 24 h). At the start of the exposure intervals (0 and 24 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure intervals (24 and 48 h), the water parameters of the old media were measured in all replicates per concentration level and control.
- Temperature: During the test period, the temperature in the incubator was 21 °C
- Dissolved oxygen concentration at the end of the exposure intervals was > 3 mg/L in the test vessels of the tested concentration levels and the control.
- pH: The deviation of the pH-value from the initial value was not more than 1.5 units.

OTHER TEST CONDITIONS
Illumination: Diffuse light; light intensity of max. 20 µE/m2/s-1
Photoperiod: 16/8 h light/dark cycle
Feeding: The daphnids were not fed during the study

EFFECT PARAMETERS MEASURED: Acute Immobilisation to Daphnia magna to determine the EC10-, EC50- and EC100-values after 24 and 48 hours of exposure under semi-static conditions.

TEST CONCENTRATIONS
- Nominal concentrations: 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L.
- Spacing factor for test concentrations: 2
- Measured concentrations: The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 80 -to 106 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 72 to 96 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be: 0.500 - 1.07 - 2.15 - 4.66 - 9.85 mg/L.
- Control: Dilution water without test item tested under the same conditions as the test groups.
- Range finding study: The test concentrations are based on the results of a non GLP preliminary range finding test under static conditions. The nominal test item concentrations in the preliminary study were 10, 1, 0.1 and 0 mg/L (control).

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL (<0.500-3.68))

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 2.66-5.01)

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

9.85 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: CL not determined

Details on results:

The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression. The EC50-value was determined to be 1.62 mg/L (CI 1.57 - 1.66 mg/L).

Results with reference substance (positive control):

The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

- Methods of evaluation: The EC100 - values after 24 and 48 h were estimated directly from the observation data, without mathematical calculation.
- EC-values and statistical analyses: The EC10- and the EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5, wherever appropriate. The concentration effect relationships are shown graphically. The EC50-value for the reference item and its confidence limits were calculated accordingly.

Description of key information

Toxic effects have been observed in a short-term toxicity test to aquatic invertebrates according to OECD 202.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

4.01 mg/L

Additional information

In the acute immobilization test with Daphnia magna, the effects of the test item were determined according to OECD 202. The study was conducted under semi-static conditions over a period of 48 h with 5 concentrations of the test item in the range of 0.625 to 10.0 mg/L, prepared with dilution water in a geometric series with a separation factor of 2.

The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 80 to 106 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 72 to 96 % of the nominal values. The geometric mean measured concentrations of the test item were calculated to be: 0.500, 1.07, 2.15, 4.66 and 9.85 mg/L. The validity criteria of the test guideline were fulfilled. Based on the geometric mean measured concentrations of the test item, the 48h-EC values were determined to be as follows: EC10 = 2.09 mg/L, EC50 = 4.01 mg/L and EC100 = 9.85 mg/L.